Clusterin调控肝癌细胞sorafenib耐药的分子机制研究

Clusterin (CLU) is multifunctional glycoprotein that plays important roles in several physiological and pathological processes. CLU also exerts a critical role in cancer development and progression. Our previous study indicated that CLU was significantly upregulated during sorafenib treatment and conferred sorafenib resistance in heaptocellular carcinoma (HCC) cell lines. However, the molecular mechanisms that CLU involved in sorafenib resistance are still unclear. In this project, we are planing to elucidate the role of CLU in the regulation sorafenib resistance in HCC cell lines using shRNA-mediated down-regulation of CLU or ectopic expression of CLU. We will also employ a tissue microarray to examine the expression of CLU and its correlation with the sensitivity of sorafenib in clinic. Given the significant role of CLU as a molecular chaperone, we will set out to identify CLU-binding partners by co-immunoprecipitation followed by nano-liquid chromatography / tandem mass spectrometry (LC-MS/MS). Protein array will be utilized to explore molecular mechanisms that how CLU confers sorafenib resistance in HCC cells. The shRNA or CRISPR-based screen thecnology will be employed to identify the functional clue of CLU-binding partners. The network regulatory relationships between CLU-binding partners and downstream signalings will be further explored. In addition, this project will investigate the feasibility and rationality of combination of sorafenib and OGX-011, an antisense oligodeoxynucleotide (ODN) targeting CLU for HCC patients. This study may provide a novel and promising therapeutic approach to the treatment of HCC.

Clusterin（CLU）作为一种多功能蛋白，与多种恶性肿瘤的发生发展密切相关。我们前期研究发现索拉菲尼处理后肝癌细胞CLU显著上调并促进肝癌细胞存活。目前关于CLU促进肝癌细胞耐药的分子机制尚不明确。本课题首先通过体内体外实验进一步确认CLU对肝癌细胞索拉菲尼耐药的影响，同时结合肝癌组织样本分析CLU表达与肝癌患者索拉菲尼敏感性之间的相关性。基于CLU作为分子伴侣的重要作用，采取蛋白质组学分析CLU的相互作用蛋白谱；通过蛋白芯片等高通量方法确定CLU调节索拉菲尼耐药的下游分子机制，同时结合高通量shRNA或CRISPR文库筛查技术分析相互作用蛋白功能，并深入研究该相互作用蛋白与下游信号通路之间的网络调控关系，解析CLU调控肝癌细胞索拉菲尼耐药的分子机制。此外，本课题还将探讨CLU的分子靶向药物OGX-011与索拉菲尼联合运用的合理性及可行性，本研究有望为肝癌的治疗提供新的实验依据。

本课题以肝癌一线药物Sorafenib为研究对象，聚焦临床面临的关键科学问题：肝癌患者对Sorafenib反应不理想，开展系列研究。. 确认热休克蛋白Clusterin对肝癌细胞Sorafenib耐药的影响，发现Sorafenib可上调Clusterin表达，Clusterin表达与肝癌患者Sorafenib敏感性具有显著相关性。基于Clusterin作为分子伴侣的重要作用，采取蛋白质组学获得Clusterin的相互作用蛋白谱；发现Clusterin的分子靶向药物OGX-011与Sorafenib联合运用的具有显著协同效应且在动物水平证实相关结果。. 同时基于高通量功能基因筛选技术的发展，我们运用基于激酶组的CRISPR-Cas9高通量功能基因筛选方法结合后续分析发现，MEK抑制剂Selumetinib联合Sorafenib对MAPK激活肝癌细胞株具有协同作用。该研究揭示了为何临床联合Sorafenib和MEK抑制剂不能取得满意疗效的原因，并为MAPK信号通路激活的肝癌患者提供了新的联合治疗策略。. 进一步分析前期工作中基于CRISPR-Cas9高通量功能基因筛选的数据，发现CDK12可能是潜在的肝癌治疗靶点。CDK12抑制剂THZ531可以与Sorafenib或Regorafenib发生协同作用，该协同作用主要来源于联合处理可以有效抑制Sorafenib单药处理所导致的细胞存活通路激活。. 综上所述，该项目系统解析了Sorafenib耐药的潜在分子机制，并从三个方面提出潜在的应对策略，为临床进展期肝癌的治疗提供了新的思路。